BR112021006578A2 - aurora kinase inhibitor for use in the treatment of neuroblastoma - Google Patents

Abstract

KINASE AURORA INHIBITOR FOR USE IN THE TREATMENT OF NEUROBLASTOMA. The present invention provides an Aurora A kinase inhibitor, Formula (I) illustrated below or a pharmaceutically acceptable salt thereof for use in the treatment of neuroblastoma.

Description

Invention Patent Descriptive Report for "INHIBITOR

FROM AURORA TO KINASE FOR USE IN THE TREATMENT OF NEUROBLASTOMA".

[001] The present invention relates to the use of an Aurora A kinase inhibitor and its salts for the treatment of neuroblastoma.

[002] Neuroblastoma is one of the most common solid tumors in children and more than 650 cases of neuroblastoma are diagnosed each year in North America. Neuroblastoma can be subdivided into two defined subsets of patients, often called low-risk and high-risk. Low-risk neuroblastoma is usually found in children under 18 months of age, with a limited burden of disease, resulting in a favorable prognosis. However, high-risk neuroblastoma usually occurs in children older than 18 months, often metastatic to bone tissue, resulting in a poor prognosis. Although advances in multimodal treatment strategies have led to better outcomes for patients with neuroblastoma, survival rates for patients in the high-risk category remain low, with less than 50% survival five years after diagnosis.

[003] High-risk neuroblastoma is associated with the MYCN gene, which encodes the proto-oncogene protein N-myc (N-MYC). Although not fully understood, N-MYC and Aurora A Kinase appear to interact and the expression and amplification of Aurora A kinase is considered to stabilize N-MYC and/or slow its degradation, which in turn would cause an increase in N-MYC levels . Michaelis, M, et al, "Aurora Kinases as Targets in Drug-Resistant Neuroblastoma Cells", PLOS One, 2014, 9(9) and 108758.

Aurora kinase inhibitors are known in the art (see, for example, PCT Patent Application Publication, WO2016/077191, which discloses the compound of Formula I (see below). The use of certain Aurora kinase inhibitors , including a selective Aurora A inhibitor alisertib and a pan Aurora inhibitor, tozasertib, have been associated with unacceptably high levels of neutropenia and other toxic effects.

[005] There is a need for new approaches and drugs to treat neuroblastoma, in particular, high-risk neuroblastoma. Furthermore, there is a need to provide methods of inhibiting Aurora kinases, in particular Aurora A kinase and decreasing N-MYC expression and/or activity. The present invention addresses these needs and provides a method of treating neuroblastoma.

[006] In one form, the present invention provides a method for treating neuroblastoma in a patient in need of treatment. Preferably, the present invention provides a method for treating high-risk neuroblastoma in a patient in need of treatment. The method comprises administering to the patient an effective amount of a compound which is (2R,4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluoro-6-acid -[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidine-4-carboxylic acid, illustrated below as Formula I, or a pharmaceutically acceptable salt of the compound of Formula I. In one embodiment, the compound of Formula I is provided as a free acid. In another embodiment, the compound of Formula I is provided as a base addition salt. In a preferred embodiment, the compound of Formula I is provided as a 2-methylpropan-2-ammonium salt (also known as an erbumine salt or a tert-butylamine salt) which is the acid ((2R, 4R)-1 -[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]- 2-methyl-piperidine-4-carboxylic acid: 2-methyl-2-propanamine (1:1)). In another embodiment, the compound of Formula I is provided as an ammonia salt of ((2R,4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluor-6] -[(5-

methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidine-4-carboxylic : amine salt (1:1)).

Formula I

[007] In another form, the present invention provides a pharmaceutical composition comprising the compound of Formula I, or its pharmaceutically acceptable salt and one or more of: pharmaceutically acceptable carrier, diluent or excipient for use in the treatment of neuroblastoma, preferably for the treatment of high-risk neuroblastoma. In one embodiment, the composition comprises a compound of Formula I, which is a free acid. In another embodiment, the composition comprises a compound of Formula I as a base addition salt, preferably a 2-methylpropan-2-ammonium salt or an ammonia salt, more preferably a methylpropan-2-ammonium salt.

[008] The present invention provides the compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of neuroblastoma. The present invention also provides the use of the compound of Formula I, or its pharmaceutically acceptable salt, for the manufacture of a medicament for the treatment of neuroblastoma. In one embodiment, the compound is provided as a free acid. In another embodiment, the compound of Formula I is provided as a base addition salt. In a preferred embodiment, the compound of Formula I is provided as a salt of 2-methylpropan-2-ammonia. In yet another embodiment, the compound of Formula I is provided as an ammonia salt.

[009] The compound of Formula I, or its pharmaceutically acceptable salt, can be used in combination with standard treatment for patients in need of treatment for neuroblastoma. Standardized treatment may include one or more of the following: surgery or excision of all or part of the tumor, radiation therapy, stem cell transplantation, administration of chemotherapeutic agents, differentiating agent, and immunotherapy.

[0010] Examples of additional chemotherapeutic agents that can be combined or administered with the compound of Formula I, or its pharmaceutically acceptable salt include: alkylators (cyclophosphamide, temozolomide and melphalan hydrochloride), platinum agents (carboplatin, cisplatin and oxaliplatin), anthracyclines (doxorubicin hydrochloride), topoisomerase I inhibitors (irinotecan and topotecan) and vinca alkaloids (vincristine sulfate). Differentiating agents include isotretinoin (13-cis-retinoic acid) and immunotherapeutic agents include monoclonal antibodies such as GD2 monoclonal antibodies (dinutuximab). The compound of Formula I or its pharmaceutically acceptable salt and one or more additional chemotherapeutic agents, differentiating agents and/or immunotherapeutic agents can be administered simultaneously, separately or sequentially to treat neuroblastoma.

The term "pharmaceutically acceptable salt", as used herein, refers to salts of the compound of Formula I. Examples of pharmaceutically acceptable salts and methods for their preparation can be found in Stahl. P, et al, "Handbook of Pharmaceutical Salts: Properties, Selection and Use", 2nd Revised Edition, Wiley-VCH, (2011) and Berge, S., M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Sciences , 1977, 66(1), 1-19; Gould, PL, "Salt selection for basic drug", International Journal of Pharmaceutics, 1986, 33: 201-217; and Bastin, RJ,

et al. "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities", Organic Process Research and Development, 2000, 4(5) 427-435.

The compound of Formula I, or a pharmaceutically acceptable salt thereof, may be formulated for administration as part of a pharmaceutical composition. Preferred pharmaceutical compositions may be formulated as a tablet or capsule for oral administration, a solution for oral administration or an injectable solution. The tablet, capsule or solution can include the compound of Formula I or a pharmaceutically acceptable salt thereof, in an amount effective to treat neuroblastoma in a patient in need of treatment. More preferably, such compositions are for oral administration. As such, pharmaceutical compositions comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, can be in combination with one or more pharmaceutically acceptable additives. The term "pharmaceutically acceptable (acceptable) additive(s)", as used herein for pharmaceutical compositions, refers to one or more of: vehicles, diluents and excipients that are compatible with the other additives in the composition or formulation and are not harmful to the patient. Examples of pharmaceutical compositions and processes for their preparation can be found in "Remington: The Science and Practice of Pharmacy", Loyd, V., et al. Eds., 22nd Ed., Mack Publishing Co., (2012). Non-limiting examples of pharmaceutically acceptable carriers, diluents and excipients include the following: saline, water, starch, sugars, mannitol and silica derivatives; binding agents such as carboxymethylcellulose, alginates, gelatin and polyvinylpyrrolidone; kaolin and bentonite; and polyethyl glycols.

"Effective amount" means the amount of the compound of Formula I or its pharmaceutically acceptable salt; or pharmaceutical composition containing the compound of Formula I, or its pharmaceutically acceptable salt, that will elicit the desired biological or medical response or therapeutic effect in a tissue, system, animal, mammal or human being sought by the researcher, veterinarian, physician or another clinician. In certain embodiments, effective amount refers to that amount of the compound of Formula I, or pharmaceutically acceptable salt thereof, that when administered is effective to delay, stop, or reverse the progression of neuroblastoma; or delay or arrest the growth or proliferation of neuroblastoma cells in a patient.

[0014] The effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof, actually administered that will elicit the desired biological or medical response or therapeutic effect in a tissue, system or patient will be determined by a physician under relevant circumstances , including the condition to be treated, the chosen route of administration, the actual compound of the present invention administered, the age, weight and response of the individual patient, and the severity of the patient's symptoms. Dosages per day typically range from about 0.1 to about 100 mg. In some cases, dosage levels below the lower limit of this range may be more than adequate, while in other cases, even higher doses may be employed. Preferred dosages are in the range of 1 to 80 mg; more preferably between 1 and 50 mg; even more preferably between 1 and 30 mg; even more preferably between 1 to 25 mg. Dosages can be administered once, twice, three times or more per day. In one embodiment, the compound of the present invention can be administered at a dosage of 15 mg or 25 mg per dose given orally twice daily (BID).

[0015] As used herein, the term "patient" refers to a human or non-human mammal. More particularly, the term "patient" refers to a human.

[0016] The term "treating" (or "treating" or "treatment") refers to the process that involves decelerating, interrupting, stopping, controlling, reducing or reversing the progression or severity of a symptom, disorder, condition or disease, such as neuroblastoma

[0017] As used herein, the following expressions have the indicated meanings: "ATCC" refers to the American Type Culture Collection; "BID" refers to twice-daily dosing; "DMEM" refers to Dulbecco's modified Eagle's medium; "DNA" refers to deoxyribonucleic acid; "EMEM" refers to Eagle's Minimum and Essential Medium; "F12" refers to the F12 middle of Ham; "FBS" refers to Fetal Bovine Serum; "HBSS" refers to Hank's Balanced Salt Solution; "HSRRB" refers to the Health Science Research Resources Bank; "JCRB" refers to the Japanese Collection of Research Bioresources; "MEM" refers to the Minimum Essential Medium; "NBL" refers to neuroblastoma; "NEAA" refers to non-essential amino acids; "PBS" refers to phosphate buffered saline; "RPMI" refers to the Roswell Park Memorial Institute; and "SCID" refers to severely matched immunodeficient mice.

The compound of Formula I and its pharmaceutically acceptable salts, including the 2-methylpropan-2-ammonium and ammonia salts, can be prepared according to the synthetic methods described in US 9,637,474. Biological Assays Monolayer Antiproliferation Assays

[0019] One measure of the potency of an Aurora A inhibitor is its ability to inhibit the proliferation of cancer cells in culture due to cell cycle disruption and mitotic catastrophe. The antiproliferative activity of Aurora A inhibitor in NBL cell lines may be indicative of clinical response to Aurora A inhibitors. NBL tumor cell lines are recovered from frozen stocks and grown for 1 to 2 passages in cell culture flasks. NBL tumor cell lines include: CHP-212, GOTO, IMR-32, NB16, NH-6, SH-SY5Y, SK-N-AS, SK-N-DZ, SK-NF 1, SK-N-MC , SK-N-SH and TGW detailed in Table 1. Table 1 Strain Catalog Supplier Lot No Histology Complete Medium Cell No. DMEM:F12(1: CHP-212 ATCC CRL-2273 58063161 neuroblastoma 1)+10% FBS EMEM + 10 % IMR32 ATCC CCL-127 59587034 neuroblastoma

FBS DMEM + 0.1 mM SK-N-AS ATCC CRL-2137 58078525 NEAA neuroblastoma + 10%

FBS neuroblastoma with alpha-MEM + NH-6 JCRB JCRB0832 06262000 opsomioclonus, 10% FBS trunk ataxia DMEM + 0.1 brain, SK-N-DZ ATCC CRL-2149 3903996 mM NEAA + neuroblastoma 10% FBS DMEM + 0.1 brain, SK -N-FI ATCC CRL-2142 58078707 mM NEAA + neuroblastoma 10% FBS brain, EMEM + 10% SK-N-SH ATCC HTB-11 59257297 neuroepithelioma FBS RPMI1640:ME GOTO HSRRB JCRB0612 neuroblastoma M(1:1) + 10%

FBS RPMI1640 + NB16 RIKEN RCB0478 neuroblastoma 15% FBS MEM:F12(1:1) SH-SY5Y ATCC CRL-2266 neuroblastoma + 10% FBS MEM + 10% TGW HSRRB JCRB0618 neuroblastoma

FBS DMEM + 0.1 KELLY Sigma 92110411 neuroblastoma mM NEAA + 10% FBS

[0020] The antiproliferative activity of an Aurora A inhibitor can be measured by the CellTiter Glo® assay. Prior to treatment with the compound of Formula I, cells are plated in complete growth medium in white-walled clear-bottom microtiter plates at a predetermined optimal density for each cell line. Sixteen hours after plating, the compound of Formula I is added. After two cell duplications after compound addition, CellTiter-Glo® reagents are prepared according to manufacturer's protocols and added to each well. Plates are incubated at room temperature for 10 minutes and then read with a luminescence plate reader according to the manufacturer's protocol for CellTiter-Glo® Luminescent Cell Viability Assay, Promega Catalog # G7571.

[0021] The antiproliferative activity of an Aurora A inhibitor can also be measured by cell count after treatment. For this assay, NBL cell lines SK-N-DZ, SK-N-F1 and KELLY are seeded in complete growth medium in black-walled clear-bottom microtiter plates at 5,000 cells per well. Sixteen hours after plating, the compound of Formula I is added over 72 hours. Cells are then fixed in 3.7% formaldehyde (Sigma # F-1268,) permeabilized with 0.1% Triton X-100 (Roche # 92522020) in PBS for 10 minutes, then the DNA is stained with Hoechst 33342 (Mol. Probes # H-21492) diluted 1:5000 in PBS. Stained plates are screened with a Celllnsight NXT® (Thermo Fischer) tracking platform using target activation bioapplication to quantify nuclei per field, a measure of cells per well. For both assays, absolute EC50 values are reported from 10-point serial dilution curves of Formula I.

As illustrated in Table 2, pediatric NBL cell lines are highly sensitive to in vitro treatment with the compound of Formula I. This indicates that the compound of Formula I may be effective in inhibiting cell growth of a variety of cell lines. of neuroblastoma cells. Table 2 Name of Replications Replications Mean Experimental Experimental Lineage Type (ABS† Cell Biological Assay Techniques IC50) CHP-212 CTG* 6 0.085 GOTO CTG 4 20,000 IMR-32 CTG 4 0.016 NB16 CTG 4 0.035 NH-6 CTG 4 0.031 SH- SY5Y CTG 4 0.047 SK-N-AS CTG 4 0.823 SK-N-DZ CTG 4 0.044 SK-N-DZ Image‡ 4 0.135 SK-N-FI CTG 4 0.098 SK-N-FI Image 4 0.290 SK-N-MC CTG 4 0.873 SK-N-SH CTG 4 0.078 TGW CTG 4 0.154 KELLY Image 4 0.396 *CTG refers to CellTiter-Glo® Luminescent Cell Viability Assay performed by HDBiosciences; † ABS means absolute mean; ‡ Image = anti-proliferation assay measured by cell counts (nuclear stain) Efficacy of Isolated Agent in Neuroblastoma Tumor Xenograft Models

[0023] The efficacy of the compound of Formula I, or a pharmaceutically acceptable salt thereof, can be evaluated in in vivo mouse models of neuroblastoma. The compound of Formula I as the salt of 2-methyl-2-propanamine (34.5 mg/kg) can be administered orally to nude mice or CB-17 SCID with cell-derived xenografts (CDX) using a dosing schedule of 28 days BID. Tumor volume and body weight can be measured twice a week.

[0024] The following protocol can be used to measure reductions in tumor volume in response to an active pharmaceutical ingredient. Human NBL cancer cells are culture-expanded, harvested in cycles, and injected into 5 x 106 cells in 200 µL of a 1:1 solution of HBSS and Matrigel® subcutaneously into the right rear flank of female mice (20-24 g, Charles River Laboratories). The following cell line/mouse strain combinations are used: SH-SY5Y (ATCC, # CRL-2226) in athymic nude mice, KELLY (Sigma- # 92110411) in CB-17 SCID and IMR-32 mice (ATCC, # CCL -127) in CB-17 SCID mice.

[0025] The compound of Formula I is formulated as the salt of 2-methyl-2-propanamine in 20% 2-hydroxypropyl-β-cyclodextrin in 25 mM phosphate buffer, pH 2 and administered orally at 34.5 mg/kg BID for 28 days. Body weight and tumor volume are measured twice a week.

The compound of Formula I as the salt of 2-methyl-2-propanamine was found to have % regression values as given in Table 3. Table 3 Evaluation of Compound of Formula I as the Salt of 2-Methyl- 2- Propanamine in Neuroblastoma Xenograft Models % of % Regression Model Change Type N (-) at End of p-value Xenograft Weight Body Treatment KELLY CDX* 5 -59.4 <0.001 -5.3 SH-SY5Y CDX 5 -78.8 <0.001 -1.1 IMR-32 CDX 4 -94.3 <0.001 4.7

CDX refers to Cell Derived Xenograft Type. N refers to # of replications

These results indicate that the compound of Formula I as the salt of 2-methyl-2-propanamine demonstrates significant antitumor activity in human NBL xenograft models. The Formula I compound as the 2-methyl-2-propanamine salt is effective as a single agent in 100% (3/3) of pediatric NBL mouse models tested in vivo, with results ranging from stable disease to complete response .

Claims (9)

1. Use of an effective amount of a compound which is (2R, 4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluoro-6-[(5-) acid -methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidine-4-carboxylic acid, or a pharmaceutically acceptable salt thereof, characterized in that it is for the manufacture of a medicine for treating neuroblastoma in a patient in need of such treatment. 2. Use according to claim 1, characterized in that the compound is (2R, 4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3- fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidine-4-carboxylic acid. 3. Use according to claim 1, characterized in that the compound is (2R, 4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3- fluor-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidine-4-carboxylic: 2-methylpropan-2-amine salt (1: 1). 4. Use according to claim 1, characterized in that the compound is the acid (2R, 4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3- fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidine-4-carboxylic: amine salt (1:1). 5. Pharmaceutical composition for the treatment of neuroblastoma, characterized in that it comprises a compound, as defined in any one of claims 1 to 4, and a pharmaceutically acceptable vehicle, diluent or excipient. 6. Use of an effective amount of a pharmaceutical composition as defined in claim 5, characterized in that it is for the manufacture of a drug for treating neuroblastoma in a patient in need of such treatment. 7. Combination for the treatment of neuroblastoma, characterized in that it comprises a compound, as defined in any one of claims 1 to 4, or a pharmaceutical composition, as defined in claim 5, with standardized treatment for patients selected from one or more of the following: surgery or excision of all or part of the tumor, radiation therapy, stem cell transplantation, administration of chemotherapeutic agents, differentiating agent, and immunotherapy. 8. A compound, as defined in any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, characterized in that it is for the manufacture of a drug for the treatment of neuroblastoma. 9. Invention, characterized by any of its embodiments or claim categories encompassed by the subject matter initially disclosed in the patent application or in its examples presented herein.